The broad objectives of this research are to characterize the biological chemistry of sulfur and selenium. Although the biological chemistry of these two essential elements has been the subject of extensive research, some reactions central to their biological chemistry have not been characterized. Also, because of the complexity of biological systems, most studies have used highly purified compounds in aqueous solution. In the proposed research, state-of-the-art high field nuclear magnetic resonance (NMR) spectroscopy and modern separation methods will be used to characterize the biological chemistry of sulfur and selenium both in intact red blood cells and in aqueous solution. A major objective of the research proposed in this application is to characterize the reversible formation of disulfide bonds in biological molecules. The reversible formation of disulfide bonds is used in biology to transport reducing equivalents, to regulate metabolism, to provide stability to proteins and as a cellular defense system. There are four major parts to the proposed research. (i) To characterize the response of intact red blood cells to oxidative stress caused by oxidation of thiol groups on the exofacial surface by thiol/disulfide exchange, and to determine the significance of this as a mechanism for reduction of plasma disulfides, including disulfides of thiol-containing drugs such as captopril and penicillamine. (ii) To develop methods for the noninvasive measurement of intracellular redox potential by NMR. Such methods will have many applications, for example to determine the response of thiol systems in tumors and other tissues to radiation and chemotherapy. (iii) To characterize the kinetics and thermodynamics of the formation and reduction of disulfide bonds in synthetic peptides, peptide hormones and neurotoxins by thiol/disulfide exchange reactions. The knowledge to be gained from these studies will contribute to the development of effective methods for the formation of disulfide bonds in peptides and proteins "engineered" for specific applications. (iv) To characterize the kinetics and thermodynamics of intracellular thiol/disulfide exchange reactions catalyzed by thioltransferase (TTase). The redox potential of the two cysteine residues at the active site of TTase will be determined and the mechanism of the enzyme catalyzed reactions will be characterized. The long term objectives of the research on the biological chemistry of selenium are to determine why nature chose selenium, rather than sulfur, for the active site of glutathione-peroxidase (GSH-Px) and to elucidate the mechanism by which inorganic selenium is incorporated into the 21st amino acid, selenocysteine. In the proposed research, the fundamental properties of selenium-containing compounds will be characterized in terms of the kinetics and thermodynamics of their redox reactions. Such fundamental information will contribute not only to our understanding of why nature chose selenium for the active site of GSH-Px but also to the possible development of selenodrugs.